In ischemic stroke, multiple types of brain cells in the neurovascular unit engage in a complex array of signaling mechanisms evolving during brain injury and leading to inflammation and cell death. The nuclear protein HMG-1 is a master regulator of inflammation. Once released from cells, HMG-1 upregulates a host of inflammatory cytokines and activates a number of different inflammatory cells. The known downstream receptor targets for HMG-1 are RAGE, TLR2 and TLR4. Very little is known, however, about whether this upstream signaling molecule HMG-1 plays a role in neuroinflammation, in particular following ischemic stroke. Preliminary data, obtained in a mouse model of transient brain ischemia, suggest that HMG-1 is both translocated from the nucleus and released from the cytosol rapidly after ischemic brain injury. We therefore hypothesize that HMG-1, once released from brain cells after ischemic stroke, triggers an inflammatory response by upregulating inflammatory mediators and recruiting leukocytes to the ischemic territory. We will explore the novel hypothesis that the inflammatory response elicited by HMG-1 contributes to a cascade of injury pathways within the neurovascular unit exacerbating ischemic damage. Blockade of HMG-1 signaling may therefore be a new and effective approach to reducing ischemic injury. We propose 3 aims to explore this hypothesis. Aim 1 will confirm and extend preliminary data establishing that brain ischemia causes HMG-1 translocation from the nucleus to the cytoplasm and then its release into the extracellular space. Aim 2 will characterize the signaling pathways of HMG-1 in brain cells, in vitro, based on results from other systems. This will include assessing the expression of HMG-1 putative receptors (RAGE, TLR2 and TLR4), as well as the up-regulation of cytokines (TNF-a, IL-1[unreadable]) and other inflammatory-associated molecules associated with stimulation of MAPK and NF?B upon treatment with recombinant HMG-1 and blockade by monoclonal anti-HMG-1, particularly within the endothelium. Aim 3 will test the hypothesis that inhibition of HMG-1 signaling modulates the inflammatory response and possibly reduces brain lesion size after MCAo in mouse. We therefore propose to explore biochemical and molecular approaches to demonstrate a pivotal role for HMG-1 in ischemic brain injury and by so doing, investigate novel signaling cascades and treatment targets for stroke. The inflammatory response to brain ischemia has been causally linked to brain damage. The nuclear protein HMG-1 has been shown to be a master regulator of inflammation in some situations like systemic sepsis, but it is not known whether it plays any role in inflammation following brain ischemia. This application proposes to explore biochemical and molecular approaches to demonstrate a pivotal role for HMG-1 in ischemic brain injury and by so doing, investigate novel signaling cascades and treatment targets for stroke. [unreadable] [unreadable] [unreadable]